The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. We have been using an in vitro approach to study events in reverse transcription and the role of the viral nucleocapsid protein (NC) in increasing the efficiency of viral DNA synthesis. We have shown that HIV-1 NC reduces reverse transcriptase (RT) pausing near the murine leukemia virus polypurine tract (PPT) during elongation of (-) strand DNA. Mutational analysis indicates that the features of HIV-1 NC which contribute to NC activity include: the basic residues flanking the N-terminal zinc finger, the zinc finger structures, and the cysteine and aromatic amino acids within both fingers. We have also developed an in vitro HIV-1 system to study the first strand-transfer, which occurs after synthesis of (-) strong stop (SS)DNA. Using donor and acceptor RNAs containing TAR (a large stem-loop in the terminal repeat region which could interfere with annealing of (-) SSDNA and acceptor), we observe that the products are largely heterogeneous (+) strand DNAs formed by a self-priming mechanism from (-) SSDNA; in this case, transfer efficiency is only 2%. Deletion of TAR or addition of NC greatly stimulates strand-transfer and drastically reduces self-priming. Thus, NC overcomes the potential inhibitory effect of TAR, while allowing the virus to maintain TAR for subsequent transactivating activity. Experiments to determine whether self-priming can be detected in endogenous reactions with HIV-1 virions are in progress. In other work, we have analyzed HIV-1 RT mutants having alanine substitutions in the "primer grip" region. These mutants can extend DNA primers, but most of the mutants cannot utilize RNA primers for initiation of (+) and (-) strand DNA synthesis; several mutants are also deficient in specific RNase H cleavage at the 3' terminus of the PPT. We conclude that the primer grip region plays a major role in recognition of primer-template structures containing the PPT or tRNALys3 RNA primers. Other studies have focused on a model system which mimics NC-mediated maturation of the genomic RNA dimer in virions. We find that the basic residues flanking the N-terminal zinc finger and the phenylalanine in the first finger are critical for NC function. Surprisingly, the conserved zinc finger structures themselves are not required.